In blood coagulation, thrombin cleaves fibrinogen (AaBbg)2, sites for fibrin polymerization are revealed, and fibrin clot formation begins. Thrombin-activated Factor XIII is responsible for catalyzing the formation of covalent cross-links between fibrin molecules and in fibrin-enzyme complexes. The main objective of the research project is to examine how fibrin clot architecture can be regulated by FXIII. Intermolecular cross-links involving the fibrinogen aC region help secure lateral fibrin aggregation and lead to production of a more robust fibrin clot. Clot character can also be altered by influencing thrombin's ability to cleave the FXIII activation peptide segment. The roles of individual aC and FXIII amino acid regions in clot formation will be assessed. A greater understanding of these molecular details can be translated into new strategies to positively impact medical conditions. The vital biomaterial fibrin can be manipulated to create a stronger or weaker blood clot. FXIII and the resultant fibrin network are already known to play crucial roles in stemming blood loss, in wound healing, and in cardiovascular disease. For the development of new therapeutics to regulate blood clot structure, critical gaps in our knowledge must be resolved. More information is needed on 1) the kinetic and structural features of fibrinogen aC, a key substrate for FXIII and 2) the use of FXIII A2 mutants to fine tune FXIII activation and its subsequent functional effects. The proposed research for this application will thus address two hypotheses: 1) FXIII is hypothesized to take advantage of specific reactive glutamines within the C (233-425) segment, along with local and more distant C residues, to promote formation of fibrin - cross-links. Such covalent cross-links enhance intermolecular fibrin chain contacts, clot strength, and protection from degradation and 2) FXIII A2 V34X mutants are hypothesized to possess different abilities to be activated and the extent of tranglutaminase function can be used to alter fibrin clot character. The FXIII A2 mutants will include the common polymorphism L34 and the aromatic substitutions F34, W34, and Y34. The biochemical methods required to address these aims include mass spectrometry, solution NMR, kinetic assays, and measurements of fibrin clot formation and architecture. The knowledge gained from these studies will be used to improve the utilization of a biomaterials-based network for therapeutic purposes.